ERα-positive Breast Cancer Research Articles

OR16-06 A Novel Pathway Controlling Anticancer Drug Induced Immunogenic Necrosis In ERα Positive Cancer Cells

Abstract Disclosure: J. Zhu: None. S. Ghosh: None. D. Duraki: None. M. Boudreau: Other; Self; Patent on ErSO. M. Jabeen: None. C. Mao: None. B. Park: None. G. Cheng: None. E.R. Nelson: None. P.J. Hergenrother: Advisory Board Member; Self; System Oncology. Other; Self; Patent on ErSO. D.J. Shapiro: Other; Self; Patent on ErSO. Acting through estrogen receptor alpha (ER), in orthotopic mouse xenograft models and a PDX, our non-competitive anticancer drug ErSO induces complete or near complete regression of primary and metastatic therapy-resistant ERα-positive breast, ovarian and endometrial tumors. ErSO is also highly effective in ovarian cancer PDOs from patient ascites. Unlike most anticancer agents which inhibit cancer cell proliferation or induce apoptosis, ErSO induces immune-cell-activating necrosis through ERα dependent sustained hyperactivation of the anticipatory unfolded protein response (a-UPR) pathway resulting in ATP depletion and cell swelling followed by membrane rupture and necrotic cell death. The medium from cancer cells killed by ErSO not only robustly activates mouse and human macrophages, but also dramatically increases monocyte migration. ErSO therefore has the potential to help extend the reach of immunotherapy to many solid tumors that do not express neoantigens. However, unlike apoptosis which has an established signaling pathway, how ErSO-induced necrosis was elusive. From a genome-wide CRISPR-Cas9 screen in MCF7 cells with negative selection against ErSO, we identified a guanine exchange factor (GEF) protein as a top target. Consistent with the screen, through subsequent knockout and overexpression of this protein in human breast cancer cells, we found that the knockout cells have significant resistance to ErSO while the overexpressing cells exhibit increased sensitivity to killing by ErSO. Our data indicates that this protein is not an upstream regulator of the ErSO-induced necrosis pathway. Instead, it is a regulator of the last stage of necrosis - cell membrane rupture. Thus, we identified a cytoskeleton-modifying signaling pathway with multiple components that plays a pivotal role in whether the breast cancer cell responds to a-UPR activation and swelling by membrane rupture and necrotic cell death or reorganizes its cytoskeleton enabling the cancer cell to survive. RNA-seq and other studies indicate that by regulating cytoskeleton organization, this axis is important in ErSO-persister breast cancer cells exhibiting long-term survival in ErSO. Unlike wild type and overexpressing cells, the knockout cells and persister cells display the persister cell phenotype - rapid re-growth of the cancer cells once ErSO is removed. This work enables identification of breast cancer patients whose elevated levels of components of this signaling pathway make them most likely to benefit from this novel therapy. Here, we describe a therapy that induces necrosis and immunogenic cell death in ERα-positive breast, ovarian and endometrial cancer cells. We show that, as cancer cells respond to this unique therapeutic approach, a cytoskeleton regulating pathway plays a critical role in life-death decisions. Presentation: Friday, June 16, 2023

Molecular characterization of breast cancer cell pools with normal or reduced ability to respond to progesterone: a study based on RNA-seq

BackgroundAbout one-third of patients with estrogen receptor alpha (ERα)-positive breast cancer have tumors which are progesterone receptor (PR) negative. PR is an important prognostic factor in breast cancer. Patients with ERα-positive/PR-negative tumors have shorter disease-free and overall survival than patients with ERα-positive/PR-positive tumors. New evidence has shown that progesterone (P4) has an anti-proliferative effect in ERα-positive breast cancer cells. However, the role of PR in breast cancer is only poorly understood. MethodsWe disrupted the PR gene (PGR) in ERα-positive/PR-positive T-47D cells using the CRISPR/Cas9 system. This resulted in cell pools we termed PR-low as P4 mediated effects were inhibited or blocked compared to control T-47D cells. We analyzed the gene expression profiles of PR-low and control T-47D cells in the absence of hormone and upon treatment with P4 alone or P4 together with estradiol (E2). Differentially expressed (DE) genes between experimental groups were characterized based on RNA-seq and Gene Ontology (GO) enrichment analyses. ResultsThe overall gene expression pattern was very similar between untreated PR-low and untreated control T-47D cells. More than 6000 genes were DE in control T-47D cells upon stimulation with P4 or P4 plus E2. When PR-low pools were subjected to the same hormonal treatment, up- or downregulation was either blocked/absent or consistently lower. We identified more than 3000 genes that were DE between hormone-treated PR-low and control T-47D cells. GO analysis revealed seven significantly enriched biological processes affected by PR and associated with G protein-coupled receptor (GPCR) pathways which have been described to support growth, invasiveness, and metastasis in breast cancer cells. ConclusionsThe present study provides new insights into the complex role of PR in ERα-positive/PR-positive breast cancer cells. Many of the genes affected by PR are part of central biological processes of tumorigenesis.

Proliferative response of ERα-positive breast cancer cells to 10 μM enterolactone, and the associated alteration in the transcriptomic landscape

Enterolactone (EL) is a product of gut-microbial metabolism of dietary plant lignans. Studies linking EL with breast cancer risk have bolstered investigations into its effects on the mammary epithelial cells, and the mechanisms thereof. While it binds to the estrogen receptor α (ERα), its effect on the proliferation of mammary tumor cell lines is reportedly ambivalent; depending on its concentration. The genomic correlates of EL actions also remain unexplored. Here we have elaborately studied the effect of EL on proliferation of ERα-positive, and ERα-negative cell lines. 10 µM EL significantly enhanced the growth of the ERα-positive MCF-7 or T47D breast cancer cells, but not the ERα-negative MDA-MB-231 or MDA-MB-453 cells. In MCF-7 cells, it significantly increased the expression of TFF1 mRNA, an estrogen-induced transcript. The binding of ERα to the estrogen response element within the TFF1 locus further demonstrated the pro-estrogenic effect of 10 µM EL. We further explored the genome-wide transcriptomic effect of 10 µM EL using the next generation sequencing technology (RNA-seq). Analysis of RNA-seq data obtained from vehicle (0.1% DMSO)- or 10 µM EL-treated MCF-7 cells revealed modulation of expression of diverse sets of functionally related genes, which reflected cell cycle progression. The manner in which 10 µM EL regulated the hallmark G2/M checkpoint, and estrogen-response-late genes correlated with proliferation inducing, and estrogen-like effects of EL on MCF-7 cells.

ERα/LKB1 complex upregulates E-cadherin expression and stimulates breast cancer growth and progression upon adiponectin exposure.

Adiponectin is the major adipocytes-secreted protein involved in obesity-related breast cancer growth and progression. We proved that adiponectin promotes proliferation in ERα-positive breast cancer cells, through ERα transactivation and the recruitment of LKB1 as ERα-coactivator. Here, we showed that adiponectin-mediated ERα transactivation enhances E-cadherin expression. Thus, we investigated the molecular mechanism through which ERα/LKB1 complex may modulate the expression of E-cadherin, influencing tumor growth, progression and distant metastasis. We demonstrated that adiponectin increases E-cadherin expression in ERα-positive 2D and higher extent in 3D cultures. This occurs through a direct activation of E-cadherin gene promoter by ERα/LKB1-complex. The impact of E-cadherin on ERα-positive breast cancer cell proliferation comes from the evidence that in the presence of E-cadherin siRNA the proliferative effects of adiponectin is no longer noticeable. Since E-cadherin connects cell polarity and growth, we investigated if the adiponectin-enhanced E-cadherin expression could influence the localization of proteins cooperating in cell polarity, such as LKB1 and Cdc42. Surprisingly, immunofluorescence showed that, in adiponectin-treated MCF-7 cells, LKB1 and Cdc42 mostly colocalize in the nucleus, impairing their cytosolic cooperation in maintaining cell polarity. The orthotopic implantation of MCF-7 cells revealed an enhanced E-cadherin-mediated breast cancer growth induced by adiponectin. Moreover, tail vein injection of MCF-7 cells showed a higher metastatic burden in the lungs of mice receiving adiponectin-treated cells compared to control. From these findings it emerges that adiponectin treatment enhances E-cadherin expression, alters cell polarity and stimulates ERα-positive breast cancer cell growth in vitro and in vivo, sustaining higher distant metastatic burden.

ERα prevents tumorigenesis of both liver and breast cancer cells through CCN5

Estrogen receptor alpha (ERα)-mediated estrogen signaling has also shown to prevent hepatic tumorigenesis in mice. Consistent with this, hormone replacement therapy with estrogen supplementation dramatically reduced the risk of hepatocellular carcinoma. Silencing of ERα is also a key event for the transformation of ERα-positive breast cancer cells into malignant triple-negative breast cancer cells. However, the mechanisms underlying ERα-mediated prevention of both hepatic and mammary tumorigenesis in humans are still unclear. Here, we present a functional genomics study of ERα targeting by comparing human liver cancer cells with human breast cancer cells using “loss or gain of function” genetic assays of ERα in vitro and in vivo. We discover that cellular communication network factor 5 (CCN5) is a direct downstream target of ERα; ERα suppresses growth and prevents tumorigenesis and malignant transformation of both liver and breast cancer cells through CCN5 in humans. The ERα-CCN5 regulatory axis functions as suppressors for both hepatic and mammary tumors, which is a common mechanism of preventing tumorigenesis for both liver cancer and breast cancer in humans.

The G-protein-coupled estrogen receptor, a gene co-expressed with ERα in breast tumors, is regulated by estrogen-ERα signalling in ERα positive breast cancer cells

GPER is a seven transmembrane G-protein-coupled estrogen receptor that mediates rapid estrogen actions. Large volumes of data have revealed its association with clinicopathological variables in breast tumors, role in epidermal growth factor (EGF)-like effects of estrogen, potential as a therapeutic target or a prognostic marker, and involvement in endocrine resistance in the face of tamoxifen agonism. GPER cross-talks with estrogen receptor alpha (ERα) in cell culture models implicating its role in the physiology of normal or transformed mammary epithelial cells. However, discrepancies in the literature have obfuscated the nature of their relationship, its significance, and the underlying mechanism. The purpose of this study was to assess the relationship between GPER, and ERα in breast tumors, to understand the mechanistic basis, and to gauge its clinical significance. We mined The Cancer Genome Atlas (TCGA)-BRCA data to examine the relationship between GPER and ERα expression. GPER mRNA, and protein expression were analyzed in ERα-positive or -negative breast tumors from two independent cohorts using immunohistochemistry, western blotting, or RT-qPCR. The Kaplan-Meier Plotter (KM) was employed for survival analysis. The influence of estrogen in vivo was studied by examining GPER expression levels in estrus or diestrus mouse mammary tissues, and the impact of 17β-estradiol (E2) administration in juvenile or adult mice. The effect of E2, or propylpyrazoletriol (PPT, an ERα agonist) stimulation on GPER expression was studied in MCF-7 and T47D cells, with or without tamoxifen or ERα knockdown. ERα-binding to the GPER locus was explored by analysing ChIP-seq data (ERP000380), in silico prediction of estrogen response elements, and chromatin immunoprecipitation (ChIP) assay. Clinical data revealed significant positive association between GPER and ERα expression in breast tumors. The median GPER expression in ERα-positive tumors was significantly higher than ERα-negative tumors. High GPER expression was significantly associated with longer overall survival (OS) of patients with ERα-positive tumors. In vivo experiments showed a positive effect of E2 on GPER expression. E2 induced GPER expression in MCF-7 and T47D cells; an effect mimicked by PPT. Tamoxifen or ERα-knockdown blocked the induction of GPER. Estrogen-mediated induction was associated with increased ERα occupancy in the upstream region of GPER. Furthermore, treatment with 17β-estradiol or PPT significantly reduced the IC50 of the GPER agonist (G1)-mediated loss of MCF-7 or T47D cell viability. In conclusion, GPER is positively associated with ERα in breast tumors, and induced by estrogen-ERα signalling axis. Estrogen-mediated induction of GPER makes the cells more responsive to GPER ligands. More in-depth studies are warranted to establish the significance of GPER-ERα co-expression, and their interplay in breast tumor development, progression, and treatment.

Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer.

e13077 Background: The past decade has seen significant advancement in increasing survival estrogen receptor alpha (ERα) positive breast cancer. The use of selective estrogen receptor down-regulators and modulators (SERMs) and cyclin-dependent kinase inhibitors aided to extend overall survival of breast cancer patients. However, resistance to endocrine therapy is a common occurrence and patients will eventually succumb to their disease. Fatty acid synthase (FASN), a key enzyme in lipid biosynthesis, is overexpressed in more aggressive and therapy-resistant tumors, including breast cancers. FASN inhibitor, TVB-2640, has been evaluated in multiple tumor cell lines and in a phase 1 clinical study, and showed partial responses in 5 patients and multiple patients with prolonged stable disease (≥16 weeks). Methods: We generated therapy-resistant cells by long term exposure to tamoxifen (MCF7/TamR cells), and fulvestrant (MCF7/FR cells). Palbociclib-resistant (MCF7/RB1Crispr and ZR75/RB1Crispr) cells were generated through CRISPR/Cas9 knockout of the retinoblastoma (RB) gene. We assessed the impact of TVB-3166 inhibitor in these cells on proliferation, viability, cell cycle, apoptosis, ERα expression in patient derived explants, and tumor growth in xenografts. RNA sequencing of tamoxifen- and fulvestrant-resistant cells was performed to investigate gene expression. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blot after treatment with TVB with or without the addition of palmitate or BAPTA. Results: TVB treatment leads to a marked inhibition of proliferation in tamoxifen-, fulvestrant- and palbociclib-resistant cells compared to the parental cells. RNA sequencing of explants of patients with ERα positive disease showed down regulation of ESR1 related genes and genes involved in invasiveness. RNA sequencing of fulvestrant-resistant cells showed that treatment with TVB results in down regulation of EMT and E2F target genes, cholesterol homeostasis genes and mTORC1 signaling. Additionally, TVB significantly inhibited tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB treatment is mediated through the induction of endoplasmic reticulum stress. Conclusions: Our preclinical data provide evidence that FASN inhibition presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered.

17β-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor-positive (ER+) BC, we hypothesized that 17β-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17β-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7's miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17β-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.

Local production of active vitamin D3 metabolites in breast cancer cells by CYP24A1 and CYP27B1

The role of vitamin D3 and its metabolites in cancer and especially as a treatment option has been widely disputed. Clinicians noting low serum 25-hydroxyvitamin D3 [25(OH)D3] levels in their patients, recommend vitamin D3 supplementation as a method of reducing the risk of cancer; however, data supporting this are inconsistent. These studies rely on systemic 25(OH)D3 as an indicator of hormone status, but 25(OH)D3 is further metabolized in the kidney and other tissues under regulation by several factors. This study examined if breast cancer cells also possess the ability to metabolize 25(OH)D3, and if so, whether the resulting metabolites are secreted locally; if this ability reflects ERα66 status; and if they possess vitamin D receptors (VDR). To address this question, estrogen receptor alpha (ERα) positive (MCF-7) and ERα negative (HCC38 and MDA-MB-231) breast cancer cell lines were examined for expression of ERα66, ERα36, CYP24A1, CYP27B1, and VDR as well as for local production of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after treatment with 25(OH)D3. The results showed that independent of ER status, breast cancer cells express the enzymes CYP24A1 and CYP27B1, which are responsible for converting 25(OH)D3 into its dihydroxylated forms. Moreover, these metabolites are produced at levels comparable to the levels observed in blood. They are positive for VDR, indicating that they can respond to 1α,25(OH)2D3, which can upregulate CYP24A1. These findings suggest that vitamin D metabolites may contribute to the tumorigenicity of breast cancer via autocrine and/or paracrine mechanisms.

Design, docking, MD simulation and in-silco ADMET prediction studies of novel indole-based benzamides targeting estrogen receptor alfa positive for effective breast cancer therapy

Breast cancer is one of the most common malignancies in women, afflicting millions of lives each year. Our current study suggests that the development of the most promising 7-substituted -1-(4-(piperidine-1-yl methoxy)benzyl)-1H-indole-3-carboxamide derivatives results in potent anticancer agents through in-silico investigations. The molecular docking was performed against estrogen receptor alpha (ER-α) positive (PDB ID: 3UUD) of breast cancer cells to anticipate the binding modes of the designed compounds and the likely mode of action. The interactions between the ligands and amino acid residues were thoroughly elucidated. The stability of the docked protein-ligand complexes was further confirmed by 100 ns molecular simulations methods. From in-silico studies, indole-based benzamides exhibited satisfactory physicochemical, drug-likeness and toxicity properties. To conclude, the most promising substituted benzamide analogs on the indole ring could serve as a possible modulator against ER-α positive breast cancer.

Reduced Expression of RBP7 is Associated with Resistance to Tamoxifen In Luminal A Breast Cancer.

Tamoxifen is the most commonly used hormonal treatment for ERα-positive breast cancer. Tamoxifen resistance is still a big problem for ERα target therapy. RBP7 is a member of the cellular retinol-binding protein family. This study aims to investigate the prognostic role of RBP7 and the relationship between RBP7 expression and sensitivity or resistance to tamoxifen in ERα-positive breast cancer. A bioinformatics method was used to investigate RBP7 expression and the prognostic value of RBP7 in different subtypes of breast cancer. The relationship between RBP7 expression and sensitivity or resistance to tamoxifen was studied using clinical data (GSE1379) and cell line data (GSE27473, GSE2645923, GSM3715281, and GSM3715282). Transfection of RBP7 experiments was used to testify to the function of RBP7 in MCF7 cell. RBP7 is a member of the family of cellular retinol-binding proteins. RBP7 expression was down-regulated at both mRNA and protein levels in breast cancer and was not associated with different TNM (Tumor, Node, Metastasis) stages. High expression of RBP7 was significantly related to good relative percent survival in the luminal A subtype, but in negative breast cancer, the result was opposite. The ROC plot showed that RBP7 had a significant predictive value for the tamoxifen response in the luminal A subtype. The expression of RBP7 from patients with recurrence treated with tamoxifen was significantly reduced. Gene Expression Omnibus showed that RBP7 expression was reduced considerably in tamoxifen-resistant MCF7 cells and T47D cells. The expression of RBP7 was positively correlated with some microRNAs involved in negatively regulating tamoxifen-resistant breast cancer. We also found that the expression of RBP7 decreased significantly in tamoxifen-resistant MCF7 cells, and transfection of RBP7 increased the sensitivity of resistant cells to tamoxifen. Reduced expression of RBP7 is associated with resistance to tamoxifen in luminal A breast cancer. Our research may help to explore the mechanisms of resistance of breast cancer to tamoxifen.

A functional genetic screen for metabolic proteins unveils GART and the de novo purine biosynthetic pathway as novel targets for the treatment of luminal A ERα expressing primary and metastatic invasive ductal carcinoma.

Targeting tumor cell metabolism is a new frontier in cancer management. Thus, metabolic pathway inhibitors could be used as anti-estrogen receptor α (ERα) breast cancer (BC) drugs. Here, the interplay among metabolic enzyme(s), the ERα levels and cell proliferation was studied. siRNA-based screen directed against different metabolic proteins in MCF10a, MCF-7 and MCF-7 cells genetically resistant to endocrine therapy (ET) drugs and metabolomic analyses in numerous BC cell lines unveil that the inhibition of GART, a key enzyme in the purine de novo biosynthetic pathway, induces ERα degradation and prevent BC cell proliferation. We report here that a reduced GART expression correlates with a longer relapse-free-survival (RFS) in women with ERα-positive BCs. ERα-expressing luminal A invasive ductal carcinomas (IDCs) are sensitive to GART inhibition and GART expression is increased in receptor-positive IDCs of high grade and stage and plays a role in the development of ET resistance. Accordingly, GART inhibition reduces ERα stability and cell proliferation in IDC luminal A cells where it deregulates 17β-estradiol (E2):ERα signaling to cell proliferation. Moreover, the GART inhibitor lometrexol (LMX) and drugs approved for clinical treatment of primary and metastatic BC (4OH-tamoxifen and the CDK4/CDK6 inhibitors) exert synergic antiproliferative effects in BC cells. In conclusion, GART inhibition by LMX or other inhibitors of the de novo purine biosynthetic pathway could be a novel effective strategy for the treatment of primary and metastatic BCs.

Abstract LB134: Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth

Abstract Background: In-frame chimeric proteins that are encoded by ESR1 exons 1 to 6 (ESR1-e6) fused to C-terminal gene partners can induce hormone-independent growth and endocrine therapy (ET) resistance of estrogen receptor-α (ERα) positive breast cancer by driving a unique transcriptional signature (PMID: 34711608). However, the mechanism how active ESR1 fusions reprogram transcription is not understood. Moreover, the lack of targeted treatment due to the loss of ligand binding domain (LBD) in ESR1 gene fusions necessitates the need to develop new therapies targeting active ESR1 fusions that promote tumorigenesis. Methods: An estrogen response element (ERE) DNA pull down assay was performed to identify coactivators recruited by active ESR1 fusions. An alamar blue assay was utilized to assess cell growth after treatment with pharmacological inhibitors to the coactivators. Single cells isolated from ERα+ patient-derived xenografts (PDX) were used to form organoids (PDxOs). A CellTiter-Glo 3D assay was conducted to measure the growth of PDxOs after treatment with coactivator inhibitors. Results: Using ERE DNA pull down assays, we identified bromodomain-containing proteins (BRDs) and steroid receptor coactivators (SRCs/p160 family members) as key regulators recruited by active ESR1 fusions from nuclear extracts of breast cancer cells. A pan-BET family inhibitor (BETi), birabresib, and pan-SRC inhibitors (SRCi), SI-2 and SI-12, inhibited the growth of ERα positive breast cancer cells and PDxOs expressing an active ESR1 fusion or LBD point mutation. Importantly, the combination of birabresib and a CDK4/6 kinase inhibitor (palbociclib) conferred the largest growth reduction of PDxOs expressing an ESR1-YAP1 fusion. RNA sequencing showed BETi and SRCi led to downregulation of ERα target gene expression and cell cycle pathways, with upregulation of apoptosis pathways. Here, we propose a model whereby BRD and SRC protein families are major regulators of active ESR1 fusion-driven gene transcription and growth and positioned the potential therapeutic significance of BETi and SRCi to be tailored to treat active ESR1-translocated breast tumors. Conclusions: Active ESR1 fusions cannot be treated with standard-of-care ET due to the lack of the LBD of ERα. Therefore, it is critical to investigate how active, in-frame ESR1 fusion proteins induce gene transcription and to develop molecular mechanism-based targeted therapies. Here, we revealed two groups of coactivators (BRDs and SRCs) as critical transcriptional regulators of active ESR1 fusion-driven tumor biology. Our study demonstrated the efficacy of a BETi (birabresib) and SRCi (SI-2/SI-12) in inhibiting the viability of active ESR1 fusion/LBD mutation expressing breast cancer cells and 3D grown PDxOs. Importantly, birabresib combined with a standard-of-care CDK4/6 inhibitor conferred the best PDxO growth reduction. Citation Format: Xuxu Gou, Craig T. Vollert, Yue Chen, Diana Fandino, Matthew V. Holt, Beom-Jun Kim, Qin Feng, Jin Wang, David M. Lonard, Bert W. O'Malley, Matthew J. Ellis, Charles E. Foulds. Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB134.

Role of Secreted Frizzled-Related Protein 1 in Early Breast Carcinogenesis and Breast Cancer Aggressiveness

A human transcriptome array on ERα-positive breast cancer continuum of risk identified Secreted Frizzled-Related Protein 1 (SFRP1) as decreased during breast cancer progression. In addition, SFRP1 was inversely associated with breast tissue age-related lobular involution, and differentially regulated in women with regard to their parity status and the presence of microcalcifications. The causal role of SFRP1 in breast carcinogenesis remains, nevertheless, not well understood. In this study, we characterized mammary epithelial cells from both nulliparous and multiparous mice in organoid culture ex vivo, in the presence of estradiol (E2) and/or hydroxyapatite microcalcifications (HA). Furthermore, we have modulated SFRP1 expression in breast cancer cell lines, including the MCF10A series, and investigated their tumoral properties. We observed that organoids obtained from multiparous mice were resistant to E2 treatment, while organoids obtained from nulliparous mice developed the luminal phenotype associated with a lower ratio between Sfrp1 and Esr1 expression. The decrease in SFRP1 expression in MCF10A and MCF10AT1 cell lines increased their tumorigenic properties in vitro. On the other hand, the overexpression of SFRP1 in MCF10DCIS, MCF10CA1a, and MCF7 reduced their aggressiveness. Our results support the hypothesis that a lack of SFRP1 could have a causal role in early breast carcinogenesis.

Discovery of highly potent proapoptotic antiestrogens in a series of androst-5,16-dienes D-modified with imidazole-annulated pendants

Heterocyclic derivatives of steroid hormones are potent anticancer agents, which are used in the chemotherapy of breast and prostate cancers. Here, we describe a novel series of androstenes, D-modified with imidazole-annulated pendants, with significant anticancer activity. Novel C17-linked imidazole-annulated heterocyclic derivatives of dehydropregnenolone acetate were synthesized by the cyclocondensation with amidines using 3β-acetoxy-21-bromopregna-5,16-dien-20-one as the substrate. The antiproliferative potency of all the synthesized compounds was evaluated against human prostate (22Rv1) and human breast (MCF7) cancer cell lines and cytochromes P450. The lead compound, imidazo[1,2-a]pyridine derivative 3h, was revealed to be a promising candidate for future anticancer drug design, particularly against ERα-positive breast cancer. Lead compound 3h was found to be selective against MCF7 cells with IC50 of 0.1 μM and to act as both a potent selective agent blocking estrogen receptor α, which is involved in the stimulation of breast cancer growth, and an effective apoptosis inducer. The potential ability of compound 3h to bind to ERα was studded using molecular docking and molecular dynamics simulation. The selectivity analysis showed that lead steroid 3h produces no effects on cytochromes P450 CYP17A1, CYP7A1, and CYP21A2.

Stabilization of estrogen receptor α by USP37 contributes to the progression of breast cancer.

Breast cancer is a major cause of cancer-related morbidity and mortality in women. Estrogen receptor-positive breast cancer accounts for roughly 70%-80% of breast tumors, and estrogen receptor alpha (ERα) has been considered as a key driver in promoting breast cancer progression. In the present study, we identified USP37 as a novel modulator in modulating ERα ubiquitination and stability. The expression of USP37 was upregulated in ERα-positive breast cancer and correlated with ERα protein level. High expression of USP37 was associated with unfavorable prognosis. USP37 depletion resulted in significantly decreased ERα protein level, ERα target genes expression as well as the estrogen response element activity in breast cancer cells. Further mechanistic study revealed the interaction between USP37 and ERα: USP37 regulated ERα signaling through modulating protein stability instead of gene expression, in which it stabilized ERα protein via inhibiting the K48-specific polyubiquitination process. Additionally, USP37 depletion led to growth inhibition and cell cycle arrest of ERα-positive breast cancer cells, which could be further rescued by ERα overexpression. Overall, our study proposed a novel post-translational mechanism of ERα in promoting breast cancer progression. Targeting USP37 may be proved to be a promising strategy for patients with ERα-positive breast cancer.

Non-Coding RNAs Modulating Estrogen Signaling and Response to Endocrine Therapy in Breast Cancer

The largest part of human DNA is transcribed into RNA that does not code for proteins. These non-coding RNAs (ncRNAs) are key regulators of protein-coding gene expression and have been shown to play important roles in health, disease and therapy response. Today, endocrine therapy of ERα-positive breast cancer (BC) is a successful treatment approach, but resistance to this therapy is a major clinical problem. Therefore, a deeper understanding of resistance mechanisms is important to overcome this resistance. An increasing amount of evidence demonstrate that ncRNAs affect the response to endocrine therapy. Thus, ncRNAs are considered versatile biomarkers to predict or monitor therapy response. In this review article, we intend to give a summary and update on the effects of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) on estrogen signaling in BC cells, this pathway being the target of endocrine therapy, and their role in therapy resistance. For this purpose, we reviewed articles on these topics listed in the PubMed database. Finally, we provide an assessment regarding the clinical use of these ncRNA types, particularly their circulating forms, as predictive BC biomarkers and their potential role as therapy targets to overcome endocrine resistance.

Abstract P3-11-07: Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer

Abstract INTRODUCTION: The past decade has seen significant advancement in increasing survival in estrogen receptor alpha (ERα) positive breast cancer. The use of selective estrogen receptor down-regulators and modulators (SERMs) (e.g. fulvestrant and tamoxifen), mTOR inhibitors (e.g. everolimus), aromatase inhibitors (AIs), and cyclin-dependent kinase inhibitors (e.g. palbociclib, ribociclib, and abemaciclib) have helped to extend overall survival of breast cancer patients. Unfortunately, resistance to endocrine therapy is a common occurrence and all patients will eventually succumb to their disease. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and is overexpressed in more aggressive and therapy-resistant tumors, including breast cancers. FASN inhibitor, TVB-2640, has been evaluated in multiple tumor cell lines and in a phase 1 clinical study, and showed partial responses in 5 patients and multiple patients with prolonged stable disease (≥16 weeks). METHODS: We generated tamoxifen- and fulvestrant-resistant MCF7 cells by long term exposure to tamoxifen (MCF7/TamR cells) and fulvestrant (MCF7/FR cells), and palbociclib-resistant (MCF7/RB1Crispr and ZR75/RB1Crispr) cells were generated through CRISPR/Cas9 knockout of the retinoblastoma (RB) gene. We assessed the impact of TVB-3166 inhibitor (and analog of TVB-2640 with slightly lower molecular weight for in vitro use) on proliferation, viability, cell cycle, and apoptosis in these cells. We evaluated the impact of TVB on proliferation and ERα expression in patient derived explants, and tumor growth in xenografts. RNA sequencing of tamoxifen- and fulvestrant-resistant cells was performed to investigate alterations in gene expression. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blotting after treatment with TVB with or without the addition of palmitate or BAPTA. RESULTS: TVB treatment leads to a marked inhibition of proliferation in tamoxifen-, fulvestrant- and palbociclib-resistant cells compared to the parental cells. RNA sequencing of explants of patients with ERα positive disease showed down regulation of ESR1 related genes and genes involved in invasiveness. RNA sequencing of fulvestrant-resistant cells showed that treatment with TVB results in down regulation of EMT and E2F target genes, cholesterol homeostasis genes and mTORC1 signaling. Additionally, TVB significantly inhibited tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB treatment is mediated through the induction of endoplasmic reticulum stress in tamoxifen-resistant cells. CONCLUSION: Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered. Citation Format: Henriette Balinda. Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-07.

BAP18 facilitates CTCF-mediated chromatin accessible to regulate enhancer activity in breast cancer.

The estrogen receptor alpha (ERα) signaling pathway is a crucial target for ERα-positive breast cancer therapeutic strategies. Co-regulators and other transcription factors cooperate for effective ERα-related enhancer activation. Recent studies demonstrate that the transcription factor CTCF is essential to participate in ERα/E2-induced enhancer transactivation. However, the mechanism of how CTCF is achieved remains unknown. Here, we provided evidence that BAP18 is required for CTCF recruitment on ERα-enriched enhancers, facilitating CTCF-mediated chromatin accessibility to promote enhancer RNAs transcription. Consistently, GRO-seq demonstrates that the enhancer activity is positively correlated with BAP18 enrichment. Furthermore, BAP18 interacts with SMARCA1/BPTF to accelerate the recruitment of CTCF to ERα-related enhancers. Interestingly, BAP18 is involved in chromatin accessibility within enhancer regions, thereby increasing enhancer transactivation and enhancer-promoter looping. BAP18 depletion increases the sensitivity of anti-estrogen and anti-enhancer treatment in MCF7 cells. Collectively, our study indicates that BAP18 coordinates with CTCF to enlarge the transactivation of ERα-related enhancers, providing a better understanding of BAP18/CTCF coupling chromatin remodeling and E-P looping in the regulation of enhancer transcription.

ERβ1 Sensitizes and ERβ2 Desensitizes ERα-Positive Breast Cancer Cells to the Inhibitory Effects of Tamoxifen, Fulvestrant and Their Combination with All-Trans Retinoic Acid.

Adjuvant endocrine therapy (AET) is the treatment of choice for early-stage estrogen receptor alpha (ERα)-positive breast cancer (BC). However, almost 40% of tamoxifen-treated cases display no response or a partial response to AET, thus increasing the need for new treatment options and strong predictors of the therapeutic response of patients at high risk of relapse. In addition to ERα, BC research has focused on ERβ1 and ERβ2 (isoforms of ERβ), the second ER isotype. At present, the impact of ERβ isoforms on ERα-positive BC prognosis and treatment remains elusive. In the present study, we established clones of MCF7 cells constitutively expressing human ERβ1 or ERβ2 and investigated their role in the response of MCF7 cells to antiestrogens [4-hydroxytamoxifen (OHΤ) and fulvestrant (ICI182,780)] and retinoids [all-trans retinoic acid (ATRA)]. We show that, compared to MCF7 cells, MCF7-ERβ1 and MCF7-ERβ2 cells were sensitized and desensitized, respectively, to the antiproliferative effect of the antiestrogens, ATRA and their combination and to the cytocidal effect of the combination of OHT and ATRA. Analysis of the global transcriptional changes upon OHT-ATRA combinatorial treatment revealed uniquely regulated genes associated with anticancer effects in MCF7-ERβ1 cells and cancer-promoting effects in MCF7-ERβ2 cells. Our data are favorable to ERβ1 being a marker of responsiveness and ERβ2 being a marker of resistance of MCF7 cells to antiestrogens alone and in combination with ATRA.